The invention relates to transgenic non-human mammalian animals being capable of expressing the human FKHL14/FOXC2 gene in its adipose tissue. The invention also relates to methods for identifying compounds useful for the treatment of medical conditions related to obesity or diabetes, said compounds being capable of stimulating expression of the human FKHL14/FOXC2 gene, or being capable of stimulating the biological activity of a polypeptide encoded by the human FKHL14/FOXC2 gene. The invention further relates to methods for identifying compounds useful for the treatment of medical conditions related to malnutrition, said compounds being capable of decreasing expression of the human FKHL14/FOXC2 gene, or being capable of decreasing the biological activity of a polypeptide encoded by the human FKHL14/FOXC2 gene.
More than half of the men and women in the United States, 30 years of age and older, are now considered overweight, and nearly one-quarter are clinically obese (Wickelgren, 1998). This high prevalence has led to increases in the medical conditions that often accompany obesity, especially non-insulin dependent diabetes mellitus (NIDDM), hypertension, cardiovascular disorders, and certain cancers. Perhaps most importantly, obesity confers a significant increased rate of mortality when compared with that of individuals of normal body weight. Obesity results from a chronic imbalance between energy intake (feeding) and energy expenditure. Energy expenditure has several major components including basal metabolism, physical activity, and adaptive (nonshivering) thermogenesis. This latter process refers to energy that is dissipated in response to changing environmental conditions, most notably exposure to cold or excessive caloric intake (so-called diet-induced thermogenesis). To better understand the mechanisms that lead to obesity and to develop strategies in certain patient populations to control obesity, we need to develop a better underlying knowledge of the molecular events that regulate the differentiation of preadipocytes and stem cells to adipocytes, the major component of adipose tissue.
Role of Adipose Tissue
The reason for existence of the adipocyte is to store energy for use during periods of caloric insufficiency. Postprandially, dietary fat is absorbed via the intestine and secreted into the circulation as large triglyceride (TG) rich particles called chylomicrons (chylo). Lipoprotein lipase (LPL), although produced by adipocytes, is localized to the endothelial cell surface where it hydrolyses TG resulting in the release of free fatty acids (FFA). Much of these are taken up by the adipose tissue either passive or active via FFA transporters. The FFAs are then activated to an acyl CoA form and re-esterfied by an enzymatic cascade to form storage TG. At the same time, glucose, which also increases in the circulation postprandially, is taken up into adipose tissue via specific plasma membrane glucose transporters. These two substrates (glucose and FFA) are the building blocks for formation of storage TG. On the other hand, during fasting, FFAs are released from the adipose tissue TG pool through the action of hormone sensitive lipase (HSL; FIG. 1). Clearly, efficient functioning of adipose tissue is dependent on the coordinated control of each of these processes and the proteins involved.
In recent years, a growing body of evidence has demonstrated a dual role for adipocytes, also being a source of numerous hormones that regulate both the adipocyte itself and many other systems within the body. Adipocytes produce leptin as a function of adipose energy stores. Leptin acts through receptors in the hypothalamus to regulate appetite, activity of brown adipose tissue (BAT), insulin secretion via sympathetic nervous system output, and important neuroendocrine adaptive responses to fasting and control of reproduction. The gene encoding leptin was identified by positional cloning (Zhang et al., 1994) and is the mutation leading to the profound obese phenotype of the ob/ob mouse, characterized by severe obesity, NIDDM, diminished fertility and hypothermia. The db-gene codes for a hypothalamic receptor for leptin (Chua et al., 1996) and the db/db mutant mice show a similar phenotype with ob/ob mice, but here the defect lies in the block of leptin receptor downstream signaling. After leptin administration, it was possible to correct the defect only in the ob/ob, but not db/db mice as predicted by Coleman""s parabiosis experiments (Coleman, 1973).
Another adipocyte product, the cytokine tumor necrosis factor xcex1 (TNFxcex1), has profound effects on adipocyte differentiation, and energy metabolism, and can even induce adipocyte dedifferentiation and apoptosis. Furthermore, TNFxcex1 has more systemic implications as it has been shown to play a role in the genesis of insulin resistance associated with obesity (Hotamisligil et al., 1993). In obese humans and numerous rodent models of obesity-diabetes syndromes, there is a marked elevation in muscle and adipose TNFxcex1 production, as compared with tissues from lean individuals (Hotamisligil et al., 1995; Hotamisligil et al., 1993). TNFxcex1 levels can be reduced with weight loss (Hotamisligil et al., 1995) or after treatment with the insulin-sensitizing agent pioglitazone (Hofmann et al., 1994).
A third adipocyte product, the acylation stimulating protein (ASP) exert autocrine action on the adipocyte, having potent anabolic effects on human adipose tissue by stimulation of glucose transport and FFA esterification (Maslowska et al., 1997; Walsh et al., 1989). ASP is generated by the interaction of complement D (identical to adipsin), factor B, and complement C3, components of the alternate complement pathway all produced by adipocytes (Choy and Spiegelman, 1996).
White Adipose Tissue Versus Brown Adipose Tissue
There are two different types of adipose tissue in the body, WAT and BAT, which have quite opposite physiological functions although they both have the same xe2x80x9cmachineryxe2x80x9d for lipogenic and lipolytic activity. WAT stores excess energy as triglycerides and releases free fatty acids in response to energy requirements at other sites. BAT on the other hand is involved in adaptive (non-shivering) thermogenesis. BAT is found only at certain sites in the body of rodent, such as in interscapular, perirenal and retroperitoneal regions. In human neonates BAT is present in large quantities but its thermogenic activity decreases shortly after birth and the tissue is gradually converted into white type adipose tissue (Lean et al., 1986). However, judged by expression of the brown fat specific uncoupling protein 1 (UCP1) mRNA, substantial amounts of brown adipocytes exist throughout life in human adipose deposits, which are generally classified as white (Krief et al., 1993).
Brown adipocytes have a multilocular disposition of fat droplets, i.e. a number of individual droplets within each adipocyte, whereas the white adipocyte has a single fat droplet within the cell. Furthermore, the brown adipocyte has a central nucleus and a large number of mitochondria in contrast to the white adipocyte, which has very few mitochondria and a nucleus that is displaced towards the plasma membrane by the lipid droplet. The only known gene marker to distinguish BAT from WAT, or any other cell types is the expression of UCP1 in brown adipocytes. Due to the presence of this unique mitochondrial protein brown adipocytes have the ability of facultative heat production, which is highly regulated by sympathetic nerve activity. UCP1 is a proton translocator in the inner mitochondrial membrane and functions as a facultative uncoupler of the mitochondrial respiratory chain (Nicholls and Locke, 1984). Recently two new uncoupling proteins have been identified and cloned through their sequence homology with UCP1. UCP2 is found in most tissues (Fleury et al., 1997), while UCP3 is expressed in BAT and skeletal muscle (Boss et al., 1997). The respective roles for UCP2 and UCP3 in thermogenesis and energy balance of intact animals remain to be determined. That brown fat is highly important in rodents for maintaining nutritional homeostasis is predicted by the facts that the function of BAT is impaired in obese rodents (Himms-Hagen, 1989) and transgenic mice with decreased brown fat mass develop obesity (Lowell et al., 1993). Since BAT is much less obvious in large animals like humans, than in rodents, skeletal muscle is thought to be the site of primary importance for normally occurring adaptive thermogenesis in large animals.
Both white and brown fat are innervated under the control of the sympathetic nervous system. There are at least three pharmacologically distinct subtypes of xcex2-adrenergic receptors (xcex21, xcex22, and xcex23) found in adipocytes. The xcex23-adrenergic receptor (xcex23-AR) is the predominant subtype in adipose tissue and it mediates the effects of norepinephrine present in the sympathetic synaptic cleft during nerve stimulation of lipolysis in WAT and BAT and of thermogenesis in BAT (Giacobino, 1995). Increased lipolysis takes place primarily through the production of cAMP and the activation of hormone-sensitive lipase through phosphorylation (FIG. 1). Thermogenesis in BAT is accomplished by increased UCP1 mRNA levels through stimulation of transcription (Rehnmark et al., 1990; Ricquier et al., 1986). Uncoupled respiration is also thought to be stimulated by increased lipolysis and the raise in intracellular concentration of FFA (Jezek et al., 1994). Sympathetic stimulation of brown fat also contributes to regulation of energy expenditure by increasing mitochondrial biogenesis (Wu et al., 1999a) and hyperplasia of brown adipocytes. In rodents, xcex23-adrenergic receptors (xcex23-ARs) are abundant in WAT and BAT (Granneman et al., 1991; Muzzin et al., 1991; Nahmias et al., 1991), while in humans, xcex23-AR mRNA is abundant in BAT only, with much less or no xcex23-AR mRNA found in WAT (Granneman and Lahners, 1994; Krief et al., 1993). Long-term treatment of obese rodents with xcex23-selective agonists reduces fat stores and improves obesity-induced insulin resistance (Bloom et al., 1992; Cawthorne et al., 1992; Holloway et al., 1992). Thus, xcex23-selective agonists are promising anti-obesity compounds. Trials of xcex23-AR agonist treatment, aimed at stimulating BAT in humans have proved disappointing with respect to weight loss (Arch and Wilson, 1996). The true potential of xcex23-AR agonists in humans can only be evaluated when a compound with good selectivity and efficacy at the human xcex23-AR, coupled with a long duration of action in vivo, has been identified, however those compounds that have been evaluated in humans so far have much lower efficacy at the human than the rodent receptor. This could be explained by the fact that human and mouse/rat xcex23-AR show a xcx9c80% similarity in their amino acid sequence. Several of the xcex23-AR-selective agonists (e.g. BRL 37344 and CL 316,243) have been shown to be extremely potent against mouse and rat xcex23-AR but with a greatly reduced activity against the human xcex23-AR. Presently, recombinant cell lines expressing human xcex23-ARs are being used to identify compounds with an increased potency against the human receptor (Ito et al., 1998). Mice with targeted mutagenesis of the xcex23-AR gene show only a modest tendency to become obese and their brown fat response to cold exposure works perfectly normal (Susulic et al., 1995). Deficient mice displayed an up-regulation of xcex21-AR mRNA levels in both white and brown fat which most probably is the reason of the mild phenotype. These results implicate that it is possible that xcex21- and xcex22-ARs also play important roles in innervation of adipose tissue. Moreover, other species, including humans, have higher levels of xcex21- and xcex22-ARs, then xcex23-AR, in adipose tissue (Lafontan and Berlan, 1993).
Adipocyte Differentiation
There has been some great progress during the past few years in the understanding of the adipocyte differentiation program. Most of the work leading to this understanding has been carried out using white preadipose cell lines in culture, notably the C3H10Txc2xd and NIH 3T3 fibroblastic cell lines and the 3T3-L1 and 3T3-F442A preadipocyte cell lines. Treatment of multipotent C3H10Txc2xd cells with 5-azacytidine (a demethylating agent) gives rise to cells committed to the myogenic, adipogenic, osteoblastic, or chondrogenic lineages. This is consistent with the view that the adipose lineage arises from the same multipotent stem cell population of mesodermal origin that gives rise to the muscle and cartilage lineages (Cornelius et al., 1994). Under appropriate hormonal control (e.g. glucocorticoid, insulin-like growth factor-1, and cyclic AMP or factors that mimic these agents) or experimental manipulation white preadipose cell lines are capable to differentiate into mature white adipocytes (Ailhaud et al., 1992). Several transcription factors have been identified, which act co-operatively and sequentially to trigger the functional differentiation program (FIG. 2).
Transcriptional Control of Adipocyte Differentiation Through PPARs C/EBPs, and ADD1/SREBP1
Peroxisome proliferator-activated receptors (PPARs) are a class of the nuclear hormone receptors. The member PPARxcex3 is now well recognized as serving an important role in the regulation of adipogenesis. Through the use of alternate promoters, the gene encoding PPARxcex3 gives rise to two separate products, PPARxcex31 and PPARxcex32, the latter containing an additional 28 N-terminal amino acids that are reported to enhance ligand binding (Fajas et al., 1997; Werman et al., 1997). Reports that ligand activation of retrovirally expressed PPARxcex32 in non-differentiating NIH-3T3 cells potently promoted adipocyte differentiation provided the most compelling evidence for the adipogenic nature of PPARxcex32 (Tontonoz et al., 1994b). One live-born PPARxcex3 deficient mouse has been produced and it displayed a total absence of WAT and BAT (complete lipodystrophy) and fatty liver, secondary to lipodystrophy (Barak et al., 1999).
Members of the PPAR family specifically function as heterodimers with the retinoid X receptor (RXR) through interactions with peroxisome proliferator response elements (PPREs) on target genes, including lipoprotein lipase (LPL; Schoonjans et al., 1996), the adipocyte fatty acid-binding protein 422/aP2 (Tontonoz et al., 1994a), phosphoenolpyruvate carboxykinase (PEPCK; Tontonoz et al., 1995), and stearocyl-CoA desaturase 1 (Miller and Ntambi, 1996). Transcriptional activity of PPARxcex3 is induced following binding of either synthetic or naturally occurring ligands, including prostaglandins of the D2 and J2 series, with the 15-deoxy-xcex94 12, 14-prostaglandin J2 derivate emerging as one of the most potent (Forman et al., 1995). Synthetic ligands that activate PPARxcex3 include carbacyclin and a new class of antidiabetic drugs, the thiazolidinediones (TZDs) (Lehmann et al., 1995). TZDs promote adipogenesis in culture and improve insulin sensitivity in vivo. PPARxcex3 activators probably modify the production of adipocyte-derived mediators of insulin resistance, such as free fatty acids or TNFxcex1. PPARxcex3 activation will decrease production of TNFxcex1 by adipocytes and interfere with its inhibitory effect on insulin signaling (Peraldi et al., 1997).
In addition, because of its tissue selective effects on genes involved in fatty acid uptake, PPARxcex3 activation will induce repartitioning of fatty acids in the body, with enhanced accumulation of fatty acids in adipose tissue at the expense of a relative depletion of muscle fatty acids (Martin et al., 1997). The relative lipid depletion of muscle cells will improve their glucose metabolism and result in an improvement in insulin sensitivity. Furthermore, PPARxcex3 decreases the expression of the adipocyte-derived signaling molecule leptin, which results in an increase in energy intake and optimization of energy usage, contributing further to PPARxcex3""s adipogenic effect (De Vos et al., 1996). Recently it has been demonstrated that interaction with a novel cofactor PPARxcex3 coactivator (PGC-1), could enhance PPARxcex3 transcriptional activity in brown adipose tissue (Puigserver et al., 1998).
Three members of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors, i.e. C/EBPxcex1, C/EBPxcex2, and C/EBPxcex4, have been implicated in the induction of adipocyte differentiation. The factors are proteins of the bZIP class, with a basic domain that mediates DNA binding and a leucine zipper dimerization domain. Cyclic AMP and adipogenic hormones such as glucocorticoids and insulin induce a transient increase in the expression of C/EBPxcex2 and xcex4 early in adipocyte differentiation (Cao et al., 1991; MacDougald et al., 1994; Yeh et al., 1995). C/EBPxcex2, in synergy with C/EBPxcex4, then induces PPARxcex3 expression in the preadipocyte (Wu et al., 1996, Wu et al., 1995). Mice lacking the C/EBPxcex2, and C/EBPxcex4 gene have normal expression of C/EBPxcex1 and PPARxcex3, but this co-expression of C/EBPxcex1 and PPARxcex3 is not sufficient for complete adipocyte differentiation in the absence of C/EBPxcex2 and C/EBPxcex4 (Tanaka et al., 1997). C/EBPxcex1 seems to play an important part in the later stages of differentiation by maintaining the differentiated adipocyte phenotype through autoactivation of its own gene (Lin and Lane, 1992; Lin and Lane, 1994). C/EBPxcex1 activates several adipocyte-specific genes such as the insulin-responsive glucose transporter-4 (GLUT4) (Kaestner et al., 1990), 422/aP2 (Christy et al., 1989), UCP1 (Yubero et al., 1994), and also the insulin receptor gene, and insulin receptor substrate 1 (IRS-1) (Wu et al., 1999b). Definitive proof that C/EBPxcex1 is required for adipocyte differentiation was obtained by showing that expression of antisense C/EBPxcex1 RNA in 3T3-L1 preadipocytes prevented differentiation (Samuelsson et al., 1991). Consistent with this finding, disruption of the C/EBPxcex1 gene gave rise to mice that failed to develop white adipose tissue (Wang et al., 1995). Taken together these findings proved that C/EBPxcex1 is both required and sufficient to induce adipocyte differentiation. The expression of C/EBPxcex1, as well as other adipocyte genes, is induced upon ligand activation of PPARxcex3. Through a positive feedback loop, C/EBPxcex1 maintains the expression of PPARxcex3. C/EBPxcex1 and PPARxcex3 cooperate to promote adipocyte differentiation, including adipocyte gene expression and insulin sensitivity (Wu et al., 1999b). It is possible that C/EBPxcex1 is ultimately an important, indirect target of the antidiabetic actions of the TZDs.
ADD1/SREBP1 (adipocyte determination and differentiation-dependent factor 1/sterol regulatory element binding protein 1) is a member of the basic helix-loop-helix (bHLH) class of transcription factors. In the inactive state, the protein is membrane-bound to the endoplasmic reticulum. Upon activation (such as a low cholesterol state), ADD1/SREBP1 is proteolytically cleaved and the soluble form becomes translocated to the nucleus where it binds one of two different response elements, namely the E box and the sterol regulatory element (SRE; Brown and Goldstein, 1997). The expression of ADD1/SREBP1 is induced during differentiation of adipocytes, where it activates transcription of target genes involved in both cholesterol metabolism and fatty acid metabolism (Kim and Spiegelman, 1996). ADD1/SREBP1 potentiates the transcriptional activity of PPARxcex3 probably through the production of endogenous ligands for PPARxcex3 (Kim et al., 1998) and also by binding to and inducing the PPARxcex3 promoter (Fajas et al., 1999).
When preadipocytes differentiate into adipocytes, several differentiation-linked genes are activated. Lipoprotein lipase (LPL) is one of the first genes induced during this process (FIG. 2). Two cis-regulatory elements important for gradual activation of the LPL gene during adipocyte development in vitro have been delimited (Enerback et al., 1992). These elements, LP-xcex1 and LP-xcex2, contained a striking similarity to a consensus sequence known to bind transcription factors of the winged helix family. Results of gel mobility shift assays and DNase I and exonuclease III in vitro protection assays indicated that factors with DNA-binding properties similar to those of the winged helix family of transcription factors are present in adipocytes and interact with LP-xcex1 and LP-xcex2. There is a need for identifying human winged helix genes that could be responsible for the induction of the LPL promoter and possibly regulating expression of other adipocyte specific genes.
xe2x80x9cFork Headxe2x80x9d and xe2x80x9cWinged Helixxe2x80x9d Genes
The xe2x80x9cfork headxe2x80x9d domain is an evolutionary conserved DNA-binding domain of 100 amino acids, which emerged from a sequence comparison of the transcription factor HNF-3xcex1 of rat and the homeotic genefork head of Drosophila. X-ray crystallography of the fork head domain from HNF-3xcex3 revealed a three-dimensional structure, the xe2x80x9ctwinged helixxe2x80x9d, in which two loops (wings) are connected on the C-terminal side of the helix-turn-helix (Brennan, R. G. (1993) Cell 74, 773-776; Lai, E. et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10421-10423).
The isolation of the mouse mesenchyme fork head-1 (MFH-1) and the corresponding human (FKHL14) chromosomal genes is disclosed by Miura, N. et al. (1997) Genomics 41, 489-492. The nucleotide sequences of the mouse MFH-1 gene and the human FKHL14 gene have been deposited with the EMBL/GenBank Data Libraries under accession Nos. Y08222 and Y08223 (SEQ ID NO: 1), respectively. The International Patent Application WO 98/54216 (published on Dec. 3, 1998) discloses a gene designated freac11, which encodes a polypeptide identical to polypeptide encoded by the human FKHL14 gene disclosed by Miura.